Treatment of kidney diseases associated with elevated AVP

ABSTRACT

Disclosed are methods for treating kidney disease including autosomal dominant polycystic kidney disease (ADPKD) in a subject, comprising the step of administering to the subject a composition comprising a therapeutically effective amount of ticagrelor or a derivative thereof, thereby treating ADPKD. Disclosed are methods of decreasing arginine vasopressin (AVP) production in a subject comprising the step of administering to the subject a composition comprising an effective amount of ticagrelor, thereby decreasing AVP production. Disclosed are methods for treating dilutional hyponatremia in a subject comprising the step of administering to the subject a composition comprising an effective amount of ticagrelor, thereby decreasing AVP production.

This patent application claims the benefit of the filing date of U.S.Ser. No. 62/452,841, filed Jan. 31, 2017, the contents of which areherein incorporated by reference in its entirety into the present patentapplication.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under IOBX000596 awardedby the U.S. Department of Veterans Affairs. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Ticagrelor (Brilinta®) has been in clinical use as an anti-clottingdrug, but not for the treatment of cystic disease. Autosomal dominantpolycystic kidney disease (ADPKD) is the most common inherited diseaseof the kidney, with a prevalence at birth ranging from 1 per 500 to 1000people worldwide. ADPKD is caused by mutations in the PKD1 (85%) or PKD2(15%) genes, which encode for polycystin-1 or polycystin-2 proteins,respectively. The hallmark of ADPKD is the formation of cysts in bothkidneys, which gradually grow in size. Over the decades, new cysts formresulting in a decline of kidney function. By the age 55 years, about50% of the ADPKD patients develop end-stage renal disease (ESRD), whichrequires dialysis therapy or renal transplantation.

There is no specific therapy for ADPKD. Its management is limited tocontrol of high blood pressure, and symptomatic treatment ofcomplications. Currently three different approaches are being tested toslow down the progression of cyst growth, but each has its ownsignificant side effects.

Therefore, finding a treatment that can be sustained long-term withoutsignificant side effects is needed. The disclosed methods provide a newtreatment for kidney diseases associated with elevated AVP (such asADPKD) with a drug that is known to be safe for long term use inpatients.

SUMMARY OF THE INVENTION

Disclosed are methods for treating kidney diseases associated withelevated AVP (such as ADPKD) in a subject, comprising the step ofadministering to the subject a composition comprising a therapeuticallyeffective amount of ticagrelor or a derivative thereof, thereby treatingthe kidney disease in the subject.

Disclosed are methods for treating autosomal dominant polycystic kidneydisease (ADPKD) in a subject, comprising the step of administering tothe subject a composition comprising a therapeutically effective amountof ticagrelor or a derivative thereof, thereby treating ADPKD, whereintreating ADPKD comprises reducing cyst number and/or size or decreasingor preventing the increase in kidney size.

Disclosed are methods for treating ADPKD in a subject, comprising thestep of administering to the subject a composition comprising atherapeutically effective amount of ticagrelor or a derivative thereof,thereby treating ADPKD, further comprising administering one or moreadditional therapeutics.

Disclosed are methods for treating a disease (such as ADPKD) associatedwith elevated AVP in a subject suffering therefrom, comprising the stepof administering to the subject a composition comprising atherapeutically effective amount of ticagrelor or a derivative thereof,thereby treating the disease in the subject.

Disclosed are methods of decreasing arginine vasopressin (AVP)production in a subject comprising the step of administering to thesubject a composition comprising an effective amount of ticagrelor,thereby decreasing AVP production.

Disclosed are methods for treating dilutional hyponatremia in a subjectcomprising the step of administering to the subject a compositioncomprising an effective amount of ticagrelor, thereby decreasing AVPproduction.

Disclosed are methods for treating ADPKD in a subject sufferingtherefrom, comprising the step of administering to the subject acomposition comprising a therapeutically effective amount of ticagreloror a derivative thereof, thereby treating ADPKD in the subject.

Disclosed are methods for inhibiting arginine vasopressin (AVP)production in hypothalamus comprising administering to hypothalamiccells or contacting hypothalamic cells with an effective amount ofticagrelor or a derivative thereof, thereby inhibiting argininevasopressin (AVP) production in hypothalamic cells.

Disclosed are methods for inhibiting arginine vasopressin (AVP)production in a subject in need thereof, comprising administering to thesubject a composition comprising an effective amount of ticagrelor or aderivative thereof, thereby decreasing AVP production in the subject.

Disclosed are methods for inhibiting cyst growth in a kidney of asubject suffering from a kidney disease (such as ADPKD) associated withelevated AVP, comprising administering to the subject a compositioncomprising an effective amount of ticagrelor or a derivative thereof,thereby inhibiting cyst growth in a kidney of the subject.

Disclosed are methods for lowering circulating levels of AVP in asubject, comprising administering to the subject an effective amount ofa composition comprising an effective amount of ticagrelor or aderivative thereof, thereby lowering circulating levels of AVP in thesubject.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 is a graph showing the effect of feeding different concentrationsof ticagrelor in the diet on plasma ticagrelor levels. (Numbers inparentheses are number of mice in each group).

FIG. 2 is a graph showing the changes in the mean urine outputs in micefed different concentrations of ticagrelor as a function of time. Thelines show the percent increase in urine outputs from day 0 to day 14.

FIG. 3 is a graph showing the changes in the mean urine osmolalities inmice fed different concentrations of ticagrelor as a function of time.The lines show the percent fall in urine osmolalities from day 0 to day14.

FIG. 4 is a graph showing the changes in the mean urine AVP excretion ingroups of mice fed different concentrations of ticagrelor in the food asa function of time. The lines show the percent fall in urine AVP withineach group from day 0 to day 14 (i.e., day 0 vs. day 14 in the samegroup).

FIGS. 5A and 5B are regression analysis graphs. A: Regression analysisshowing significant relationship between plasma ticagrelor and urineosmolality in the mice. B: Regression analysis showing significantrelationship between plasma ticagrelor and urinary AVP excretion in themice.

FIGS. 6A-C show the effect of ticagrelor on the mRNA expression of AQP2(A) and AQP3 (B) relative to the expression β-actin and the effect ofticagrelor on the mRNA expression of AQP2 relative to GAPDH (C) inprimary cultures of rat inner medullary collecting duct (IMCD) cells.Cells were incubated with different concentrations of ticagrelor (0.5 to25 μM) for 48 hours. During the second 24 hours, the cells werechallenged with dDAVP (20 nM). The numbers in parentheses above the barsindicate the number of culture wells. Statistical values are shown inthe tables.

FIG. 7 is a graph showing the effect of ticagrelor on dDAVP-induced cAMPproduction in primary cultures of rat IMCD cells: Cells were incubatedfor 48 h with ticagrelor (5 μM). During the second 24 hours, cells werechallenged with dDAVP (20 nM). N=3 Transwell® inserts for condition.*significantly different (P<0.01) when compared to CNT or Tica groups.

FIGS. 8A and 8B are graphs showing the results of cytotoxicity assays.A: LDH Cytotoxicity Assay: Effect on different concentrations ofticagrelor on the release of LDH by primary cultures of rat IMCD cellsinto the culture medium. Higher LDH release indicates increasedcytotoxicity. Results are mean±SE of 6 wells for each concentration ofticagrelor. B: Cell Cytotoxicity Assay: Effect on differentconcentrations of ticagrelor on cell cytotoxicity. Higher ratio ofA550/A605 indicates more viable cells in the well. Results are mean±SEof 6 wells for each concentration of ticagrelor.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a derivative of ticagrelor is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules are discussed, each and every combination and permutation ofderivative and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.

Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean a range of ±1-10%.

The use of the singular includes the plural unless specifically statedotherwise. The word “a” or “an” means “at least one” unless specificallystated otherwise. The use of “or” means “and/or” unless statedotherwise. The meaning of the phrase “at least one” is equivalent to themeaning of the phrase “one or more.” Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Thus, for example, reference to “a therapeutic”includes a plurality of such therapeutics; reference to “thetherapeutic” is a reference to one or more therapeutics known to thoseskilled in the art, and so forth.

The use of the term “containing,” as well as other forms, such as“contains” and “contained,” is not limiting. Also, terms such as“element” or “component” encompass both elements or componentscomprising one unit and elements or components comprising more than oneunit unless specifically stated otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety.

The term “therapeutic” refers to a composition that treats a disease.For example, the therapeutics disclosed herein are compositions thattreat autosomal dominant polycystic kidney disease.

As used herein, the term “subject” or “patient” refers to any organismto which a composition of this invention may be administered, e.g., forexperimental, diagnostic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as non-human primates, and humans;avians; domestic household or farm animals such as cats, dogs, sheep,goats, cattle, horses and pigs; laboratory animals such as mice, ratsand guinea pigs; rabbits; fish; reptiles; zoo and wild animals).Typically, “subjects” are animals, including mammals such as humans andprimates; and the like.

As used herein, the term “therapeutically effective amount” means anamount of a therapeutic, prophylactic, and/or diagnostic agent (e.g.,ticagrelor) that is sufficient, when administered to a subject sufferingfrom or susceptible to a disease, disorder, and/or condition, to treat,alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of the disease, disorder, and/or condition. In some instances,a therapeutically effective amount is an amount of a therapeutic thatprovides a therapeutic benefit to an individual.

As used herein, the term “treating” refers to partially or completelyalleviating, ameliorating, relieving, delaying onset of, inhibiting orslowing progression of, reducing severity of, and/or reducing incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition. For example, “treating” a kidney disease, e.g.,autosomal dominant polycystic kidney disease, may refer to slowing downthe progression of the disease, reducing the number or size of cysts,and/or reducing the size of the kidneys. Treatment can be administeredto a subject who does not exhibit signs of a disease, disorder, and/orcondition and/or to a subject who exhibits only early signs of adisease, disorder, and/or condition for the purpose of decreasing therisk of developing pathology associated with the disease, disorder,and/or condition.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g.,ticagrelor) and whose structure is sufficiently similar to thosedisclosed herein and based upon that similarity, would be expected byone skilled in the art to exhibit the same or similar activities andutilities as ticagrelor, or to induce, as a precursor, the same orsimilar activities and utilities as ticagrelor. Exemplary derivativesinclude salts, esters, amides, salts of esters or amides, and N-oxidesof a parent compound.

As used herein, the term “production of arginine vasopressin or AVP”refers to or encompasses transcription of AVP gene or processing of themRNA or translation or post-translational modification or storage orsecretion of a combination of one of more of these.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range¬from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise.

Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another, specifically contemplated embodiment that should beconsidered disclosed unless the context specifically indicatesotherwise. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, andindependently of the other endpoint unless the context specificallyindicates otherwise. Finally, it should be understood that all of theindividual values and sub-ranges of values contained within anexplicitly disclosed range are also specifically contemplated and shouldbe considered disclosed unless the context specifically indicatesotherwise. The foregoing applies regardless of whether in particularcases some or all of these embodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

Methods for Treating Kidney Diseases

Disclosed are methods for treating kidney diseases including thoseassociated with elevated AVP (such as ADPKD) in a subject, comprisingthe step of administering to the subject a composition comprising atherapeutically effective amount of ticagrelor or a derivative thereof,thereby treating the kidney disease in the subject. In an embodiment,ADPKD is associated with mutation of PKD1 gene and/or PKD2 gene. Inanother embodiment, ADPKD is associated with altered expression of PKD1gene and/or PKD2 gene. In another embodiment, ADPKD is associated withreduced expression of PKD1 gene and/or PKD2 gene.

As used herein, “elevated” (such as elevated AVP) means a level higherthan found in normal patients (patients without a kidney disease). Inthe case of elevated AVP, in one embodiment, the determination of anelevated AVP may be based on comparison of measured plasma AVP level ofa subject to the mean plasma AVP level and its standard deviation for areference normal population. See for example, van Londen, L. et al.(1997) Neuropsychoparmacology 17(4):284-292 for reference values relatedto measurements for a normal control population (e.g., Table 2 on page287). In a separate embodiment, elevated AVP may be based on comparisonof measured urinary AVP level of a subject collected over a period (suchas 24 hours) to the mean urinary AVP level and its standard deviationcollected over the same duration for a reference normal population.

In one embodiment, a subject is said to have an elevated AVP if theplasma AVP level of the subject is at least one standard deviation fromthe mean plasma AVP value (for example, determined in pg/ml; mean plasmaAVP value plus one standard deviation) for a normal control population.In another embodiment, the subject is said to have an elevated AVP ifthe plasma AVP level of the subject is greater than the mean plasma AVPvalue for a normal control population by at least 1.5 standarddeviations. In another embodiment, the subject is said to have anelevated AVP if the plasma AVP level of the subject is greater than themean plasma AVP value for a normal control population by at least twostandard deviations. In another embodiment, the subject is said to havean elevated AVP if the plasma AVP level of the subject is greater thanthe mean plasma AVP value for a normal control population by at least2.5 standard deviations. In another embodiment, the subject is said tohave an elevated AVP if the plasma AVP level of the subject is greaterthan the mean plasma AVP value for a normal control population by atleast three standard deviations. In another embodiment, the subject issaid to have an elevated AVP if the plasma AVP level of the subject isgreater than the mean plasma AVP value for a normal control populationby at least 3.5 standard deviations. In another embodiment, the subjectis said to have an elevated AVP if the plasma AVP level of the subjectis greater than the mean plasma AVP value for a normal controlpopulation by at least four standard deviations. In another embodiment,the subject is said to have an elevated AVP if the plasma AVP level ofthe subject is greater than the mean plasma AVP value for a normalpopulation and is between 2.5 and 6 standard deviations from the meanplasma AVP value for a normal control population.

In one embodiment, a subject is said to have an elevated AVP if theurinary AVP level of the subject is at least one standard deviation fromthe mean urinary AVP value (mean plus one standard deviation) for anormal control population. In another embodiment, the subject is said tohave an elevated AVP if the urinary AVP level of the subject is greaterthan the mean urinary AVP value for a normal control population by atleast 1.5 standard deviations. In another embodiment, the subject issaid to have an elevated AVP if the urinary AVP level of the subject isgreater than the mean urinary AVP value for a normal control populationby at least two standard deviations. In another embodiment, the subjectis said to have an elevated AVP if the urinary AVP level of the subjectis greater than the mean urinary AVP value for a normal controlpopulation by at least 2.5 standard deviations. In another embodiment,the subject is said to have an elevated AVP if the urinary AVP level ofthe subject is greater than the mean urinary AVP value for a normalcontrol population by at least three standard deviations. In anotherembodiment, the subject is said to have an elevated AVP if the urinaryAVP level of the subject is greater than the mean urinary AVP value fora normal control population by at least 3.5 standard deviations. Inanother embodiment, the subject is said to have an elevated AVP if theurinary AVP level of the subject is greater than the mean urinary AVPvalue for a normal control population by at least four standarddeviations. In another embodiment, the subject is said to have anelevated AVP if the urinary AVP level of the subject is greater than themean urinary AVP value for a normal population and is between 2.5 and 6standard deviations from the mean urinary AVP value for a normal controlpopulation.

In a separate embodiment, ADPKD is associated with reduced or alteredactivity of PKD1 protein and/or PKD2 protein. In an embodiment, ADPKD isassociated with increased renal epithelial cell proliferation. Inanother embodiment, ADPKD is associated with bilateral renal enlargementand cyst.

In an embodiment, treating kidney disease comprises inhibiting AVPproduction in the subject. In an embodiment, inhibiting AVP productionlowers AVP plasma level in the subject. In a separate embodiment,lowered AVP plasma level in the subject may be detected as a lowerurinary AVP concentration or excretion by the subject.

In some instances, treating ADPKD comprises slowing down the progressionof the disease. In an embodiment, treating kidney disease comprisesslowing down progression of the cystic disease. Slowing down theprogression of the disease does not eliminate the disease but it canresult in the subject not developing kidney failure and/or end-stagerenal disease during their lifetime. In an embodiment, slowing downprogression of the disease or of the cystic disease comprises reducingrisk of developing kidney failure and/or end-stage renal disease. Inanother embodiment, slowing down progression of the disease or of thecystic disease comprises preventing an increase in the number of renalcysts, increase in size of renal cyst, and/or increase in size or massof one or both kidneys. In another embodiment, slowing down progressionof the disease or of the cystic disease comprises reducing number ofrenal cysts, reducing size of renal cyst, and/or reducing size or massof one or both kidneys.

In an embodiment, treating kidney disease comprises reducing cAMPproduction in renal collecting duct cells of the subject. In anembodiment, the renal collecting duct cell is or comprises a principalcell. In an embodiment, treating kidney disease increases urine outputand/or decreases urine osmolarity. In an embodiment, treating kidneydisease inhibits proliferation of renal epithelial cell.

In an embodiment, treating a disease or a kidney disease associated withelevated AVP in a subject comprises lowering level of circulating AVP inthe subject.

In an embodiment, the subject is a mammal. In an embodiment, the mammalis a human, non-human primate, rabbit, sheep, rat, dog, cat, pig, ormouse. In an embodiment, the subject is a human, non-human primate,rabbit, sheep, rat, dog, cat, pig, or mouse. Non-human primate includesbut not limited to monkey, chimpanzee, gorilla, ape, lemur, macaque andgibbon. In a preferred embodiment, the non-human primate is a monkey ora chimpanzee. In some instances, the subject is a human. In someinstances, the subject has been diagnosed with a need for treatment ofADPKD prior to the administering step. The subject may be in need oftreatment of a kidney disease. Thus, the disclosed methods can, in someinstances, further comprise the step of identifying a subject in need oftreatment of ADPKD. Identifying a subject in need of treatment of ADPKDcan comprise ultrasound, CT, or MRI scans to check for kidneyabnormality or blood tests to analyze known genetic defects related toADPKD.

Disclosed are methods for treating autosomal dominant polycystic kidneydisease (ADPKD) in a subject, comprising the step of administering tothe subject a composition comprising a therapeutically effective amountof ticagrelor or a derivative thereof, thereby treating ADPKD, whereintreating ADPKD comprises reducing cyst number and/or size or decreasingor preventing the increase of kidney size. Other symptoms of ADPKD canbe affected during treatment with the disclosed compositions. Othertreatable symptoms include, but are not limited to, size of abdomen,presence of kidney stones, high blood pressure, blood in the urine,urinary tract infections, gradual decrease in kidney function, and backand neck pain.

In an embodiment, the subject is free of a coagulation disorder. In anembodiment, the coagulation disorder is a hypercoagulation disorder orthrombophilia. In an embodiment, the hypercoagulation disorder orthrombophilia is inherited hypercoagulable condition. In an embodiment,the inherited hypercoagulable condition is associated with and mayinclude any of factor V Leiden mutation, prothrombin gene mutation,antithrombin III deficiency, protein C deficiency, protein S deficiency,elevated homocysteine level, elevated fibrinogen level,dysfibrinogenemia, elevated factor VIII level, factor XIII mutation,elevated factor IX level, elevated factor XI level, fibrinolysisdisorder, plasminogen deficiency and elevated plasminogen activatorinhibitor (PAI-1).

In an embodiment, coagulation disorder is a bleeding disorder. In anembodiment, the bleeding disorder is congenital. The congenital bleedingdisorder may include any of hemophilia, factor II deficiency, factor Vdeficiency, factor VII deficiency, factor X deficiency, factor XIdeficiency, factor XII deficiency, factor XIII deficiency, vonWillebrand's disease, Bernard-Soulier syndrome, complete plasminogenactivator inhibitor 1 (PAI-1) deficiency, congenital afrinogenemia,glycoprotein VI deficiency, gray platelet syndrome, Noonan syndrome,prekallikrein deficiency, prothrombin deficiency, Stormorken syndrome,thrombocytopenia-absent radius (TAR) syndrome and Wiskott-Aldrichsyndrome.

In an embodiment, diagnosis of ADPKD comprises one or more of largeechogenic kidneys without distinct macroscopic cysts at 50% risk forADPKD, presence of bilateral renal enlargement and cysts, PKD1 genemutation, PKD2 gene mutation, and mutation in modifiers of PKD1expression or PKD2 expression.

In an embodiment, treating ADPKD comprises reducing cyst number or sizeor decreasing kidney size. In an embodiment, the method furthercomprises ameliorating one or more symptoms associated with ADPKD. In anembodiment, one or more symptoms associated with ADPKD may include anyof acute loin pain, haematuria, ballotable kidneys, sub arachnoidhemorrhage (berry aneurysm), hypertension, associated liver cyst, uremiadue to renal failure, anemia due to CKD, increase RBC or erythropoeitinsecretion.

The disclosed methods can further comprise administering one or moreadditional therapeutics. Thus, in some instances, disclosed are methodsfor treating ADPKD in a subject, comprising the step of administering tothe subject a composition comprising a therapeutically effective amountof ticagrelor or a derivative thereof, thereby treating ADPKD, furthercomprising administering one or more additional therapeutic. In someinstances, the one or more additional therapeutic is a mTOR inhibitor,such as but not limited to, Sirolimus, Everolimus (RAD001), Temsirolimus(CCI-779), Ridaforolimus (AP23573, MK-8669), Deforolimus, Dactolisib,BGT226, SF1126, PKI-587, Sapanisertib (INK128), AZD8055 and AZD2014. Ina preferred embodiment, the mTOR inhibitor may include any of Sirolimusand Everolimus. In some instances, the one or more additionaltherapeutic is a somatostatin analogue, such as but not limited to,Octreotide, Pasireotide (SOM230), dopastatin BIM-23A387, dopastatinBIM-23A760, somatostatin octapeptide-doxorubicin RC-121, somatostatinoctapeptide-doxorubicin RC-160, somatostatin octapeptide-2-pyrrolino-DOXconjugate AN-201, AN-238 (AN-201 linked to RC-121), JF-10-81,90Y-DOTATOC, 177Lu DOTATATE [177Lu]DOTA-Tyr(3)-octreotate andLanreotide. In a preferred embodiment, the somatostatin analogue mayinclude any of Octreotide and Lanreotide. In some instances, the one ormore additional therapeutic is a vasopressin V2 receptor antagonist,such as but not limited to, OPC-31260, Lixivaptan, Mozavaptan,Satavaptan, Lixivaptan, Conivaptan (YM-087), SR-121463A, VPA-985 andTolvaptan (OPC-41061). In a preferred embodiment, the vasopressin V2receptor antagonist may include any of OPC-31260 and Tolvaptan. In someinstances, the one or more additional therapeutic is an epidermal growthfactor receptor (EGFR) inhibitor, such as but not limited to, bosutinib,gefitinib, lapatinib, cetuximab, panitumumab, vandetanib, neratinib,necitumumab, osimertnib and erlotinib. In a preferred embodiment, theepidermal growth factor receptor (EGFR) inhibitor may include any ofbosutinib, gefitinib and erlotinib. A combination of mTOR inhibitors,somatostatin analogues, vasopressin V2 receptor antagonists and EGFRinhibitors can be used. In some instances, the one or more additionaltherapeutic can be administered in the same composition or in a separatecomposition as the ticagrelor. In some instances, the additionaltherapeutic can be administered simultaneously or consecutively with theticagrelor.

In an embodiment, the one or more additional therapeutic is administeredconcurrently or sequentially with ticagrelor or its derivative. In anembodiment, the one or more additional therapeutic is administeredbefore, after or taken with a composition comprising ticagrelor or itsderivative. In an embodiment, the composition comprising ticagrelor orits derivative is administered orally, intravenously, subcutaneously orintramuscularly, as an implant or patch, or via a needle ormicroneedles. In one embodiment, the one or more additional therapeuticis administered orally, intravenously, subcutaneously orintramuscularly, or as an implant or patch, or via a needle ormicroneedles.

In one embodiment, one or more additional therapeutic is administered bythe same route as a composition comprising ticagrelor or its derivative.In another embodiment, one or more additional therapeutic isadministered by a different route as a composition comprising ticagreloror its derivative. Disclosed are methods for treating ADPKD in a subjectcomprising the step of administering to the subject a compositioncomprising a therapeutically effective amount of ticagrelor or aderivative thereof, thereby treating ADPKD, wherein the step ofadministering to the subject a composition comprising an effectiveamount of ticagrelor is a long-term treatment regimen. A long-termtreatment regimen or chronic treatment regimen means a course oftreatment that lasts longer than 1 month to a life-time depending on thesubject. Typically, in a laboratory or experimental animal, such as amouse, long-term treatment regimen or chronic treatment regimen in sucha subject may be measured in months, such as lasting longer than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Typically, in human subjectsor patients, long-term treatment regimen or chronic treatment regimenmay be measured in years, such as lasting longer than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, or 30 years. In some instances, the long-termtreatment regimen lasts a lifetime.

In an embodiment, the step of administering to the subject a compositioncomprising an effective amount of ticagrelor is a long-term treatmentregimen. In an embodiment, the long-term treatment regimen is at least 2weeks. In a different embodiment, the long-term treatment regimen is atleast 1 month. In another embodiment, the long-term treatment regimen isat least 1 year. In another embodiment, the long-term treatment regimenis at least 5 years. In a further embodiment, the long-term treatmentregimen is a lifetime from a diagnosis of need to treat.

In an embodiment, the long-term treatment is continuous. In a differentembodiment, the long-term treatment is discontinuous, wherein thetreatment is interrupted by one or more period in which administrationof the composition comprising an effective amount of ticagrelor or itsderivative is withheld.

In an embodiment, withholding a composition comprising an effectiveamount of ticagrelor or its derivative is for a sufficient amount oftime or for a specified amount of time. In another embodiment,withholding a composition comprising an effective amount of ticagreloror its derivative is for a sufficient amount of time in whichwithholding inhibits or reverses uncontrolled bleeding from the wound orinternal ulcer. In an embodiment, withholding a composition comprisingan effective amount of ticagrelor or its derivative for a specifiedamount of time reduces risk of uncontrolled bleeding associated with aprocedure or therapy. In an embodiment, the procedure is surgery orintervention radiology. In an embodiment, the therapy increases bleedingtime or decreases clotting time so as to place the subject at risk foruncontrolled bleeding.

In an embodiment, decrease in AVP production is determined by comparingAVP levels detected in the blood or urine of the subject beforeadministering ticagrelor or its derivative to AVP levels detected in theblood or urine of the subject after administering ticagrelor or itsderivative. In an embodiment, AVP levels detected in the blood or urineof the subject after administering ticagrelor are detected at least oneweek after administering ticagrelor.

In some instances, the dose of tricagrelor can be 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150 mg twice per day. In some instances, thesame daily dose is received in a once per day form. In some instances,tricagrelor can be taken daily, weekly, or monthly.

Methods for Treating Disease Associated with Elevated AVP

Disclosed are methods for treating for treating a disease associatedwith elevated AVP in a subject suffering therefrom, comprising the stepof administering to the subject a composition comprising atherapeutically effective amount of ticagrelor or a derivative thereof,thereby treating the disease in the subject.

In an embodiment, the disease may include any of hypertension,preeclampsia, congestive heart failure, cardiorenal syndrome, cirrhosisof liver, diabetic ketoacidosis, post-traumatic stress disorder (PTSD),countering effect of loop diuretics, high altitude pulmonary edema,autism, syndrome of inappropriate antidiuretic hormone (SIADH),autosomal dominant polycystic kidney disease (ADPKD), dilutionalhyponatremia and disease associated with elevated activity of AVP-V2receptor-cAMP axis. In some instances, autism may be autism spectrumdisorder (ASD).

In an embodiment, elevated activity of AVP-V2 receptor-cAMP axiscomprises elevated circulating AVP and/or elevated V2 receptor signalingin the subject. In an embodiment, elevated V2 receptor signalingcomprises elevated cAMP level in a V2 receptor positive cell of thesubject.

In an embodiment, V2 receptor positive cell is a renal collecting ductcell. In an embodiment, the renal collecting duct cell comprises aprincipal cell. In an embodiment, the renal collecting duct cell is aprincipal cell. In an embodiment, the renal collecting duct cell is arenal epithelial cell. In an embodiment, the renal epithelial cell is aprincipal cell of the collecting duct. In an embodiment, the principalcell translocates aquaporin protein to apical plasma membrane. In aseparate embodiment, the aquaporin protein is in a subapical vesicleprior to transport to apical plasma membrane. In an embodiment, theprincipal cell increases expression of aquaporin gene. Aquaporin may beaquaporin protein 2 (AQP2) and aquaporin protein 3 (AQP3).

In an embodiment, translocation of aquaporin and/or increased expressionof aquaporin gene alters transepithelial water transport. In anembodiment, alteration of transepithelial water transport comprisesincreased re-absorption of water by the principal cell or renalcollecting duct cell, decreased urine output, increased urineosmolarity, and/or increased urinary AVP excretion. In an embodiment,increased urinary AVP excretion positively correlates with an increasedplasma AVP level.

In an embodiment, treating the disease in the subject comprisesinhibiting AVP production in hypothalamus. In another embodiment,treating the disease in the subject comprises lowering circulating levelof AVP in the subject. In an embodiment, lowering circulating level ofAVP reduces signaling by AVP-dependent V2 receptor in a cell of thesubject. In an embodiment, reducing signaling by AVP-dependent V2receptor decreases cAMP levels in the cell of the subject. In anotherembodiment, lowering circulating level of AVP reduces signaling byAVP-dependent V1 receptor in a cell of the subject. In an embodiment,reducing signaling by AVP-dependent V1 receptor decreases intracellularcalcium in a cell of the subject. V1a receptor may be V1a receptor orV1b receptor. In an embodiment, reducing signaling by AVP-dependent V1or V2 receptor slows or reverses a disease associated with elevated AVPin the subject.

In an embodiment, the cell of the subject is a renal collecting ductcell. In an embodiment, the renal collecting duct cell may be a renalepithelial cell. In an embodiment, the renal collecting duct cell is orcomprises a principal cell.

In an embodiment, the principal cell has a lower cAMP level. In anembodiment, the renal collecting duct cell has a lower cAMP level. In anembodiment, the renal epithelial cell has a lower cAMP level. Such alower cAMP may be due to lower plasma level of AVP, such that secondmessenger signaling through production of cAMP by AVP-dependent receptoris decreased. In a preferred embodiment, AVP-dependent receptor inprincipal cell, renal epithelial cell or renal collecting duct cell isV2 receptor or vasopressin V2 receptor.

In an embodiment, lowering of cAMP level results in a decrease number ofaquaporin proteins on apical surface of the principal cell. In anembodiment, lowering of cAMP level results in a decreased expression ofaquaporin genes. In an embodiment, aquaporin proteins may include any ofaquaporin protein 2 (AQP2) and aquaporin protein 3 (AQP3). In apreferred embodiment, aquaporin protein is aquaporin protein 2 (AQP2).

In an embodiment, decreased number of aquaporin proteins on apicalsurface of the principal cell result in a decreased re-absorption ofwater by the principal cell or renal collecting duct cell, increasedurine output, decreased urine osmolarity, and/or decreased urinary AVPexcretion. In an embodiment, decreased urinary AVP excretion positivelycorrelates with a decreased plasma AVP level.

In an embodiment, treating a disease or a kidney disease associated withelevated AVP in a subject comprises lowering level of circulating AVP inthe subject. In an embodiment, the subject may be in need of treatmentof a kidney disease.

In an embodiment, the subject is free of a coagulation disorder. In anembodiment, the coagulation disorder may be a hypercoagulation disorderor thrombophilia. In an embodiment, the hypercoagulation disorder orthrombophilia may be inherited hypercoagulable condition. In anembodiment, the inherited hypercoagulable condition may be associatedwith and may include any of factor V Leiden mutation, prothrombin genemutation, antithrombin III deficiency, protein C deficiency, protein Sdeficiency, elevated homocysteine level, elevated fibrinogen level,dysfibrinogenemia, elevated factor VIII level, factor XIII mutation,elevated factor IX level, elevated factor XI level, fibrinolysisdisorder, plasminogen deficiency and elevated plasminogen activatorinhibitor (PAI-1).

In an embodiment, coagulation disorder may be a bleeding disorder. In anembodiment, the bleeding disorder may be congenital. the congenitalbleeding disorder may include any of hemophilia, factor II deficiency,factor V deficiency, factor VII deficiency, factor X deficiency, factorXI deficiency, factor XII deficiency, factor XIII deficiency, vonWillebrand's disease, Bernard-Soulier syndrome, complete plasminogenactivator inhibitor 1 (PAI-1) deficiency, congenital afrinogenemia,glycoprotein VI deficiency, gray platelet syndrome, Noonan syndrome,prekallikrein deficiency, prothrombin deficiency, Stormorken syndrome,thrombocytopenia-absent radius (TAR) syndrome and Wiskott-Aldrichsyndrome.

In an embodiment, the method further comprises ameliorating one or moresymptoms associated with ADPKD. In an embodiment, one or more symptomsassociated with ADPKD may include any of acute loin pain, haematuria,ballotable kidneys, sub arachnoid hemorrhage (berry aneurysm),hypertension, associated liver cyst, uremia due to renal failure, anemiadue to CKD, increase RBC or erythropoeitin secretion.

In an embodiment, the method further comprises administering one or moreadditional therapeutic.

In an embodiment, the one or more additional therapeutic may be a mTORinhibitor. In an embodiment, mTOR inhibitor may include any ofSirolimus, Everolimus (RAD001), Temsirolimus (CCI-779), Ridaforolimus(AP23573, MK-8669), Deforolimus, Dactolisib, BGT226, SF1126, PKI-587,Sapanisertib (INK128), AZD8055 and AZD2014. In a preferred embodiment,mTOR inhibitor may include any of Sirolimus and Everolimus (RAD001).

In an embodiment, the one or more additional therapeutic may be asomatostatin analogue. In an embodiment, the somatostatin analogue mayinclude any of Octreotide, Pasireotide (SOM230), dopastatin BIM-23A387,dopastatin BIM-23A760, somatostatin octapeptide-doxorubicin RC-121,somatostatin octapeptide-doxorubicin RC-160, somatostatinoctapeptide-2-pyrrolino-DOX conjugate AN-201, AN-238 (AN-201 linked toRC-121), JF-10-81, ⁹⁰Y-DOTATOC, 177Lu DOTATATE[177Lu]DOTA-Tyr(3)-octreotate and Lanreotide. In a preferred embodiment,the somatostatin analogue may include any of Octreotide and Lanreotide.

In an embodiment, the one or more additional therapeutic is avasopressin V2 receptor antagonist. In an embodiment, the vasopressin V2receptor antagonist may include any of OPC-31260, Lixivaptan,Mozavaptan, Satavaptan, Lixivaptan, Conivaptan (YM-087), SR-121463A,VPA-985 and Tolvaptan (OPC-41061). In a preferred embodiment, thevasopressin V2 receptor antagonist may include any of OPC-31260 andTolvaptan.

In an embodiment, the one or more additional therapeutic may be anepidermal growth factor receptor inhibitor. In an embodiment, theepidermal growth factor receptor inhibitor may include any bosutinib,gefitinib, lapatinib, cetuximab, panitumumab, vandetanib, neratinib,necitumumab, osimertnib and erlotinib. In a preferred embodiment, theepidermal growth factor receptor inhibitor may include any of bosutinib,gefitinib and erlotinib.

In an embodiment, the one or more additional therapeutic may beadministered concurrently or sequentially with ticagrelor or itsderivative. In an embodiment, the one or more additional therapeutic maybe administered before, after or taken with a composition comprisingticagrelor or its derivative. In an embodiment, the compositioncomprising ticagrelor or its derivative may be administered orally,intravenously, subcutaneously or intramuscularly, as an implant orpatch, or via a needle or microneedles. In one embodiment, the one ormore additional therapeutic may be administered orally, intravenously,subcutaneously or intramuscularly, or as an implant or patch, or via aneedle or microneedles.

In one embodiment, one or more additional therapeutic may beadministered by the same route as a composition comprising ticagrelor orits derivative. In another embodiment, one or more additionaltherapeutic may be administered by a different route as a compositioncomprising ticagrelor or its derivative.

In an embodiment, the step of administering to the subject a compositioncomprising an effective amount of ticagrelor may be a long-termtreatment regimen. In an embodiment, the long-term treatment regimen maybe at least 2 weeks. In a different embodiment, the long-term treatmentregimen may be at least 1 month. In another embodiment, the long-termtreatment regimen may be at least 1 year. In another embodiment, thelong-term treatment regimen is at least 5 years. In a furtherembodiment, the long-term treatment regimen may be a lifetime from adiagnosis of need to treat.

In an embodiment, the long-term treatment is continuous. In a differentembodiment, the long-term treatment is discontinuous, wherein thetreatment may be interrupted by one or more period in whichadministration of the composition comprising an effective amount ofticagrelor or its derivative is withheld.

In an embodiment, withholding a composition comprising an effectiveamount of ticagrelor or its derivative may be for a sufficient amount oftime or for a specified amount of time. In another embodiment,withholding a composition comprising an effective amount of ticagreloror its derivative may be for a sufficient amount of time in whichwithholding inhibits or reverses uncontrolled bleeding from the wound orinternal ulcer. In an embodiment, withholding a composition comprisingan effective amount of ticagrelor or its derivative for a specifiedamount of time may reduce risk of uncontrolled bleeding associated witha procedure or therapy. In an embodiment, the procedure may be surgeryor intervention radiology. In an embodiment, the therapy increasesbleeding time or decreases clotting time so as to place the subject atrisk for uncontrolled bleeding.

In an embodiment, decrease in AVP production may be determined bycomparing AVP levels detected in the blood or urine of the subjectbefore administering ticagrelor or its derivative to AVP levels detectedin the blood or urine of the subject after administering ticagrelor orits derivative. In an embodiment, AVP levels detected in the blood orurine of the subject after administering ticagrelor may be detected atleast one week after administering ticagrelor.

In some instances, the dose of tricagrelor can be 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150 mg twice per day. In some instances, thesame daily dose may be received in a once per day form. In someinstances, tricagrelor can be taken daily, weekly, or monthly.

Methods of Decreasing Arginine Vasopressin (AVP) Production

Disclosed are methods of decreasing arginine vasopressin (AVP)production in a subject comprising the step of administering to thesubject a composition comprising an effective amount of ticagrelor,thereby decreasing AVP production. In some instances, the decrease inAVP production can be determined by comparing AVP levels detected in theblood or urine of the subject before administering ticagrelor to AVPlevels detected in the blood or urine of the subject after administeringticagrelor. AVP levels in the urine can correlate to the levels in theplasma, therefore using urine or blood samples for detection of AVPlevels can be appropriate.

In some instances, AVP levels detected in the blood or urine of thesubject after administering daily doses of ticagrelor can be detected atleast one week after administering ticagrelor. In some instances, AVPlevels can be detected, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days after administering ticagrelor. In some instances, AVP levels canbe detected 2, 4, 6, 7, 8, 10, or 12 weeks after administering dailydoses of ticagrelor.

The method may include methods disclosed elsewhere in the application.

Methods for Treating Dilutional Hyponatremia

Hyponatremia is a disorder characterized by an excess of body waterrelative to body content of sodium. Hyponatremia is the most commonelectrolyte disorder encountered in hospitalized patients (25-30%),associated with increased morbidity and mortality. Hyponatremia alsorepresents a huge economic burden on the health care system, typicallycosting $10,000 more per hospitalized patient, over other hospitalizedpatients that do not have hyponatremia. It is often seen in patientswith congestive heart failure (CHF), cirrhosis of liver and in SIADH(syndrome of inappropriate anti-diuretic hormone secretion) amongothers. In these conditions, the plasma levels of AVP areinappropriately high.

Disclosed are methods for treating dilutional hyponatremia in a subjectcomprising the step of administering to the subject a compositioncomprising an effective amount of ticagrelor, thereby decreasing AVPproduction.

Disclosed are methods for treating dilutional hyponatremia in a subjectcomprising the step of administering to the subject a compositioncomprising an effective amount of ticagrelor, thereby decreasing AVPproduction, further comprising ameliorating one or more symptomsassociated with dilutional hyponatremia. In some instances, one or moresymptom associated with dilutional hyponatremia can be, but is notlimited to fatigue, headache, muscle spasms, muscle cramps, confusion orhallucination.

Disclosed are methods for treating dilutional hyponatremia in a subjectcomprising the step of administering to the subject a compositioncomprising an effective amount of ticagrelor, thereby decreasing AVPproduction, further comprising administering an additional therapeutic.In some instances, the additional therapeutic is tolvaptan, a knownvasopressin V2 receptor antagonist for treating hyponatremia. In someinstances, the additional therapeutic can be administered in the samecomposition or in a separate composition. In some instances, theadditional therapeutic can be administered simultaneously orconsecutively with the ticagrelor.

In an embodiment, the method further comprises ameliorating one or moresymptoms associated with dilutional hyponatremia. The method may furthercomprise administering an additional therapeutic, in addition to acomposition comprising an effective amount of ticagrelor or a derivativethereof. In an embodiment, the additional therapeutic is a vasopressinV2 receptor antagonist. In an embodiment, vasopressin V2 receptorantagonist may include any of OPC-31260, Lixivaptan, Mozavaptan,Satavaptan, Lixivaptan, Conivaptan (YM-087), SR-121463A, VPA-985 andTolvaptan (OPC-41061). In a preferred embodiment, the vasopressin V2receptor antagonist is tolvaptan.

In an embodiment, the subject is a mammal. In an embodiment, the mammalis a human, non-human primate, rabbit, sheep, rat, dog, cat, pig, ormouse. Non-human primate includes but not limited to monkey, chimpanzee,gorilla, ape, lemur, macaque and gibbon. In a preferred embodiment, thenon-human primate is a monkey or a chimpanzee.

Methods of Reducing One or More Symptoms Associated with ADPKD

Disclosed are methods of reducing one or more symptoms associated withADPKD in a subject, comprising the step of administering to the subjecta composition comprising an effective amount of ticagrelor or aderivative thereof, so as to reduce circulating levels of AVP, therebyreducing one or more symptoms associated with ADPKD in the subject.

In an embodiment, one or more symptoms associated with ADPKD may includeany of acute loin pain, haematuria, ballotable kidneys, sub arachnoidhemorrhage (berry aneurysm), hypertension, associated liver cyst, uremiadue to renal failure, anemia due to CKD, increase RBC or erythropoeitinsecretion.

In an embodiment, the method further comprises administering one or moreadditional therapeutic. The additional therapeutic may include any of amTOR inhibitor, a somastostatin analogue, a vasopressin V2 receptorantagonist and an epidermal growth factor receptor inhibitor. Examplesof such therapeutics are provided elsewhere in the application. In anembodiment, the additional therapeutic may include any of Sirolimus,Everolimus (RAD001), Temsirolimus (CCI-779), Ridaforolimus (AP23573,MK-8669), Deforolimus, Dactolisib, BGT226, SF1126, PKI-587, Sapanisertib(INK128), AZD8055, AZD2014, Octreotide, Pasireotide (SOM230), dopastatinBIM-23A387, dopastatin BIM-23A760, somatostatin octapeptide-doxorubicinRC-121, somatostatin octapeptide-doxorubicin RC-160, somatostatinoctapeptide-2-pyrrolino-DOX conjugate AN-201, AN-238 (AN-201 linked toRC-121), JF-10-81, ⁹⁰Y-DOTATOC, 177Lu DOTATATE[177Lu]DOTA-Tyr(3)-octreotate, Lanreotide, OPC-31260, Lixivaptan,Mozavaptan, Satavaptan, Lixivaptan, Conivaptan (YM-087), SR-121463A,VPA-985 and Tolvaptan (OPC-41061), bosutinib, gefitinib, lapatinib,cetuximab, panitumumab, vandetanib, neratinib, necitumumab, osimertniband erlotinib. In a preferred embodiment, the additional therapeutic mayinclude any of Sirolimus, Everolimus (RAD001), Octreotide, Lanreotide,OPC-31260, Tolvaptan, bosutinib, gefitinib and erlotinib. In a morepreferred embodiment, the additional therapeutic is tolvaptan.

In an embodiment, the subject is a mammal. The mammal may be a human,non-human primate, rabbit, sheep, rat, dog, cat, pig, or mouse. Examplesof non-human primate include but not limited to monkey, chimpanzee,gorilla, ape, lemur, macaque and gibbon. In a preferred embodiment, thenon-human primate is a monkey or a chimpanzee. In some instances, thesubject is a human. In an embodiment, the subject is a human in need ofa treatment. In an embodiment, the subject is a human in need of atreatment for symptoms associated with ADPKD or elevated plasma AVPlevel.

Methods for Treating ADPKD

Disclosed are methods for treating ADPKD in a subject sufferingtherefrom, comprising the step of administering to the subject acomposition comprising a therapeutically effective amount of ticagreloror a derivative thereof, thereby treating ADPKD in the subject.

In an embodiment, the subject does not have an elevated level of plasmaAVP and/or elevated level of urinary AVP. In an embodiment, the subjectis free of an elevated level of plasma AVP and/or elevated level ofurinary AVP. The subject may be a human, non-human primate, rabbit,sheep, rat, dog, cat, pig, or mouse.

In an embodiment, administering to the subject a composition comprisinga therapeutically effective amount of ticagrelor or a derivative thereofcomprises reducing cyst number and/or size or decreasing or preventingthe increase of kidney size.

In an embodiment, administering to the subject a composition comprisinga therapeutically effective amount of ticagrelor or a derivative thereofalleviates or reduces one or more symptoms associated with ADPKD. In anembodiment, the one or more symptoms associated with ADPKD may includeany of acute loin pain, haematuria, ballotable kidneys, sub arachnoidhemorrhage (berry aneurysm), hypertension, associated liver cyst, uremiadue to renal failure, anemia due to CKD, increase RBC or erythropoeitinsecretion. In another embodiment, the one or more symptoms associatedwith ADPKD may include any of size of abdomen, presence of kidneystones, high blood pressure, blood in the urine, urinary tractinfections, gradual decrease in kidney function, and back and neck pain.

In an embodiment, the method further comprises administering one or moreadditional therapeutic. The additional therapeutic may include any of amTOR inhibitor, a somastostatin analogue, a vasopressin V2 receptorantagonist and an epidermal growth factor receptor inhibitor. Suchinhibitors are described elsewhere in the application.

In an embodiment, the additional therapeutic may include any ofSirolimus, Everolimus (RAD001), Temsirolimus (CCI-779), Ridaforolimus(AP23573, MK-8669), Deforolimus, Dactolisib, BGT226, SF1126, PKI-587,Sapanisertib (INK128), AZD8055, AZD2014, Octreotide, Pasireotide(SOM230), dopastatin BIM-23A387, dopastatin BIM-23A760, somatostatinoctapeptide-doxorubicin RC-121, somatostatin octapeptide-doxorubicinRC-160, somatostatin octapeptide-2-pyrrolino-DOX conjugate AN-201,AN-238 (AN-201 linked to RC-121), JF-10-81, 90Y-DOTATOC, 177Lu DOTATATE[177Lu]DOTA-Tyr(3)-octreotate, Lanreotide, OPC-31260, Lixivaptan,Mozavaptan, Satavaptan, Lixivaptan, Conivaptan (YM-087), SR-121463A,VPA-985 and Tolvaptan (OPC-41061), bosutinib, gefitinib, lapatinib,cetuximab, panitumumab, vandetanib, neratinib, necitumumab, osimertniband erlotinib. In a preferred embodiment, the additional therapeutic mayinclude any of Sirolimus, Everolimus (RAD001), Octreotide, Lanreotide,OPC-31260, Tolvaptan, bosutinib, gefitinib and erlotinib. In a morepreferred embodiment, the additional therapeutic may be tolvaptan.

In an embodiment, treating ADPKD in the subject with a compositioncomprising a therapeutically effective amount of ticagrelor or aderivative thereof, may be a long-term treatment regimen or chronictreatment regimen.

In an embodiment, the long-term treatment regimen is at least 2 weeks.In a different embodiment, the long-term treatment regimen is at least 1month. In another embodiment, the long-term treatment regimen is atleast 1 year. In another embodiment, the long-term treatment regimen isat least 5 years. In a further embodiment, the long-term treatmentregimen is a lifetime from a diagnosis of need to treat.

In an embodiment, the long-term treatment may be continuous. In adifferent embodiment, the long-term treatment may be discontinuous,wherein the treatment may be interrupted by one or more period in whichadministration of the composition comprising an effective amount ofticagrelor or its derivative is withheld.

In an embodiment, withholding a composition comprising an effectiveamount of ticagrelor or its derivative may be for a sufficient amount oftime or for a specified amount of time.

In an embodiment, ADPKD in a subject suffering therefrom may be treatedwith a composition comprising a therapeutically effective amount ofticagrelor or a derivative thereof irrespective of coagulation status.

In a separate embodiment, ADPKD in a subject suffering therefrom may betreated with a composition comprising a therapeutically effective amountof ticagrelor or a derivative thereof taking into considerationcoagulation status.

In an embodiment, the subject may be free of a coagulation disorder. Inan embodiment, the coagulation disorder may be a hypercoagulationdisorder or thrombophilia. In an embodiment, the hypercoagulationdisorder or thrombophilia may be inherited hypercoagulable condition. Inan embodiment, the inherited hypercoagulable condition may be associatedwith and may include any of factor V Leiden mutation, prothrombin genemutation, antithrombin III deficiency, protein C deficiency, protein Sdeficiency, elevated homocysteine level, elevated fibrinogen level,dysfibrinogenemia, elevated factor VIII level, factor XIII mutation,elevated factor IX level, elevated factor XI level, fibrinolysisdisorder, plasminogen deficiency and elevated plasminogen activatorinhibitor (PAI-1).

In an embodiment, coagulation disorder may be a bleeding disorder. In anembodiment, the bleeding disorder may be congenital. The congenitalbleeding disorder may include any of hemophilia, factor II deficiency,factor V deficiency, factor VII deficiency, factor X deficiency, factorXI deficiency, factor XII deficiency, factor XIII deficiency, vonWillebrand's disease, Bernard-Soulier syndrome, complete plasminogenactivator inhibitor 1 (PAI-1) deficiency, congenital afrinogenemia,glycoprotein VI deficiency, gray platelet syndrome, Noonan syndrome,prekallikrein deficiency, prothrombin deficiency, Stormorken syndrome,thrombocytopenia-absent radius (TAR) syndrome and Wiskott-Aldrichsyndrome.

In an embodiment, the subject has a hypercoagulation disorder orthrombophilia and treatment with tricagrelor may treat both ADPKD andits symptoms as well as may reduce or alleviate hypercoagulationdisorder or thrombophila. In an embodiment, the hypercoagulationdisorder or thrombophilia may be inherited hypercoagulable condition. Inan embodiment, the inherited hypercoagulable condition may be associatedwith and may include any of factor V Leiden mutation, prothrombin genemutation, antithrombin III deficiency, protein C deficiency, protein Sdeficiency, elevated homocysteine level, elevated fibrinogen level,dysfibrinogenemia, elevated factor VIII level, factor XIII mutation,elevated factor IX level, elevated factor XI level, fibrinolysisdisorder, plasminogen deficiency and elevated plasminogen activatorinhibitor (PAI-1).

In some instances, the dose of tricagrelor may be 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150 mg twice per day. In some instances, thesame daily dose may be received in a once per day form. In someinstances, tricagrelor can be taken daily, weekly, or monthly.

Methods of Decreasing Proliferation of Medullary Collecting Duct Cells

Disclosed are methods of decreasing proliferation of medullarycollecting duct cells comprising administering to a subject acomposition comprising an effective amount of ticagrelor, therebydecreasing proliferation of medullary collecting duct cells. Increasingconcentrations of ticagrelor causes a decrease in the proliferation ofmedullary collecting duct cells.

Compositions and Administration

Disclosed are compositions comprising ticagrelor and derivativesthereof. Examples of derivatives of ticagrelor can be found in U.S. Pat.Nos. 6,525,060 and 6,251,910, both of which are incorporated herein byreference. An examples of a ticagrelor derivatives can beTriazolo[4,5-D]Pyrimidine compounds.

Examples of ticagrelor and derivatives thereof can include compoundshaving the formula:

wherein: R¹ is C₃₋₅ alkyl optionally substituted by one or more halogenatoms; R² is a phenyl group, optionally substituted by one or morefluorine atoms; R³ and R⁴ are both hydroxy;R is XOH, where X is CH₂, OCH₂CH₂ or a bond; or a pharmaceuticallyacceptable salt or solvate thereof, or a solvate of such a salt providedthat: when X is CH₂ or a bond, R¹ is not propyl; when X is CH₂ and R¹ isCH₂CH₂CF₃, butyl or pentyl, the phenyl group at R² must be substitutedby fluorine; when X is OCH₂CH₂ and R¹ is propyl, the phenyl group at R²must be substituted by fluorine.

Additional examples of derivatives of ticagrelor, include, but are notlimited to,[1R-[1α,2α,3β(1R*,2S*),5β]]-3-[7-[[2-(4-Fluorophenyl)cyclopropyl]amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(hydroxymethyl)-cyclopentane-1,2-diol;[1R-[1α,2α,3β(1R*,2S*),5β]]-3-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(hydroxymethyl)-cyclopentane-1,2-diol;[1S-(1α,2α,3β(1S*,2R*),5β]]-3-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-cyclopentane-1,2-diol;1R-[1α,2α,3β(1R*,2S*),5β]]-3-[5-(Butylthio)-7-[[2-(3,4-difluorophenyl)cyclopropyl]amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(hydroxymethyl)-cyclopentane-1,2-diol;[1S-[1α,2β,3β,4α(1S*,2R*)]]-4-[5-(Butylthio)-7-[[2-(4-fluorophenyl)cyclopropyl]amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentane-1,2,3-triol;[1S-(1α,2α,3β(1S*,2R*),5β]-3-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-cyclopentane-1,2-diol;[1S-[1α,2α,3β,5β(1S*,2R*)]]-3-(2-Hydroxyethoxy)-5-[7-(2-phenylcyclopropyl)amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentane-1,2-diol[1S-[1α,2β,3β,4α(1S*,2R*)]]-4-[5-(Butylthio)-7-[[2-(3,4-difluorophenyl)cyclopropyl]amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]cyclopentane-1,2,3-triol;[1S-[1α,2α,3β(1S*,2R*),5β]]-3-[5-(Butylthio)-7-[(2-phenylyclopropyl)amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxethoxy)-cyclopentane-1,2-diol;or pharmaceutically acceptable salts or solvates thereof, or solvates ofsuch salts.

Alkyl groups, whether alone or as part of another group are straightchained and fully saturated.

Additional examples of derivatives of ticagrelor, include, but are notlimited to,[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[7-[[2-(4-Fluorophenyl)cyclopropyl]amino]-5-(propylsulphonyl)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[7-[[2-(4-Fluorophenyl)cyclopropyl]amino]-5-[(3,3,3-trifluoropropy)lthio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl)-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]-6-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[3aR-(3aα,4α,6α,6aα)]-6-[7-Amino-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-(3aα,4α,6α,6aα)]-[[6-[7-Amino-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol]oxy]aceticacid, methyl ester;[3aR-(3aα,4α,6α,6aα)]-[[6-[7-Bromo-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol]oxy]aceticacid, methyl ester;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-[[6-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yl]oxy]aceticacid, methyl ester;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[[7-[2-(3,4-Difluorophenyl)cyclopropyl]amino-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yl]oxy]-ethanol;[3aR-(3aα,4α,6α,6aα)]-6-[7-Amino-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[3aR-(3aα,4α,6aα)]-6-[7-Amino-5-(propylsulfonyl)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[3aR-(3aα,4α,6α,6aα)]-6-[7-Amino-5-(butylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol;[3aR-(3aα,4α,6α,6aα)]-6-[7-Amino-5-(butylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol,acetate;[3aR-(3aα,4α,6α,6aα)]-6-[7-Bromo-5-(butylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol,acetate;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[5-(Butylthio)-7-[[2-(3,4-difluorophenyl)cyclopropyl]amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-methanol,acetate;[3aR-[3aα,4α,6α,6aα(1S*,2R*)]]-6-[7-[[(4-Fluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimdin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-[3aα,4α,6α,6aα(1S*,2R*)]]-6-[[7-[(4-Fluorophenyl)cyclopropyl]amino]-5-(propylsulphonyl)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-[3aα,4α,6α,6aα(1S*,2R*)]]-6-[7-[[(4-Fluorophenyl)cyclopropyl]amino]-5-(butylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[1S-(1α,2α,3β(1S*2R*),5β]-3-[7-[[2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylsulphonyl)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl)-5-(2-hydroxyethoxy)-cyclopentane-1,2-diol;(1S-cis)2-[[4-[7-Chloro-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-2-cyclopenten-1-yl]oxy]-aceticacid, ethyl ester; [1S-(cis)]2-[[4-[7-Amino-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-2-cyclopenten-1-yl]oxy]-aceticacid, ethyl ester; [1S-(cis)]2-[[4-[7-Amino-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-2-cyclopenten-1-yl]oxy]-1-ethanol;[3aR-(3aα,4α,6α,6aα)]-2-[6-[7-Amino-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yloxy]ethanol;[3aR-(3aα,4α,6α,6aα)]-2-[6-[7-Bromo-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]-pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yloxy]ethanol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]-2-[6-(7-Phenylcyclopropyl)amino]-5-[(3,3,3-trifluoropropyl)thio]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-1,3-dioxol-4-yloxy]ethanol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]-6-[[7-[(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[[7-[(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylsulfonyl)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-6-[5-(Butylthio)-7-[[2-(3,4-difluorophenyl)cyclopropyl]amino]-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-ol;[3aR-[3aα,4α,6α(1R*,2S*),6aα]]-2-[6-[[5-(Butylthio)-7-chloro-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yl]oxy]-ethanol;[3aR-[3aα,4α,6aα(1R*,2S*),6aα]]-2-[6-[[5-(Butylthio)-7-[2-phenylcyclopropyl]amino-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxol-4-yl]oxy]-ethanol.

Methods of making ticagrelor and derivatives of ticagrelor can be foundin U.S. Pat. Nos. 6,525,060 and 6,251,910, both of which areincorporated herein by reference for their teaching of methods of makingticagrelor and derivatives of ticagrelor.

Derivatives of ticagrelor of particular interest are those that decreaseor lower AVP production by the hypothalamus but lacks anti-plateleteffect with no or very little effect on coagulation. Also, of interestare derivatives of ticagrelor that decrease or lower hypothalamic AVPproduction but is not a P2Y12 receptor antagonist. Decreasing orlowering hypothalamic AVP production may occur at the level of AVP geneexpression and processing of preproprotein to produce mature vasopressin(AVP) protein and may include secretion of AVP.

The disclosed compositions can be used in any of the disclosed methods.

The disclosed compositions can be pharmaceutical compositions comprisingticagrelor. That is, a pharmaceutical composition can be providedcomprising a therapeutically effective amount of ticagrelor and apharmaceutically acceptable carrier.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In the methods described herein, administration or delivery of thetherapeutics to a subject can be via a variety of mechanisms. Forexample, the therapeutic can be formulated as a pharmaceuticalcomposition.

Pharmaceutical compositions can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated.

Preparations of parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for optical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids, or binders may be desirable. Some of the compositionscan be administered as a pharmaceutically acceptable acid- orbase-addition salt, formed by reaction with inorganic acids such ashydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acidssuch as formic acid, acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleicacid, and fumaric acid, or by reaction with an inorganic base such assodium hydroxide, ammonium hydroxide, potassium hydroxide, and organicbases such as mono-, di-, trialkyl and aryl amines and substitutedethanolamines.

Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for treatingADPKD, the kit comprising a composition comprising ticagrelor andinstructions for how to administer the composition to a subject fortreatment of ADPKD.

Also disclosed are kits for treating ADPKD, the kit comprising acomposition comprising derivatives of ticagrelor and instructions forhow to administer the composition to a subject for treatment of ADPKD.

EXAMPLES Example 1

Materials and Methods:

The study evaluated the effect of increasing doses of ticagrelor from0.05% to 0.25% (incremental doses of 0.05, 0.10, 0.15, 0.20 and 0.25%)administered to mice for 2 weeks and compared them with that obtained byadministering increasing doses of clopidogrel bisulfate (20, 40 or 80mg/kg bw/day). For this study adult B6D2 mice were used. Different dosesof ticagrelor were administered embedded in diet. Clopidogrel bisulfate(Plavix® tablets) were powdered and dissolved in drinking water foradministration. The concentration of clopidogrel in the water wasadjusted on daily basis based on the water consumption by mice on theprevious day.

Rodent diets containing ticagrelor at different concentrations wereprepared as follows. 15 grams of ticagrelor was shipped to MPBiomedicals (Solon, Ohio, USA) for custom preparation of 10 kg of rodentchow in pellet form containing 0.15% ticagrelor. The custom-made dietwas received and was stored at +4° C. Later in the study, for thepreparation of diets containing 0.20% or 0.25%, the abovecustom-prepared diet (containing 0.15% ticagrelor) was used as a baseand the required amount of ticagrelor was added. Diets containing 0.05and 0.10% ticagrelor were prepared de novo.

Clopidogrel bisulfate was administered orally by mixing finely powderedPlavix® tablets (Bristol-Myers Squibb, New York, N.Y.) in drinkingwater. The concentration of the drug in the drinking water was adjusteddaily based on the water consumption of the animals on the previous day.Three different doses (20, 40 and 80 mg/kg bw/day) were administered.

Since it is not possible to work with more than 15 to 20 mice inmetabolic cages at any given time, the experimental animals were handledin two batches in a staggered fashion. Urine samples were collected onday 0 (prior to the experimental period), on days 6/7 (end of the firstweek) and days 13/14 (end of the second week). Terminal blood sampleswere collected from the posterior vena cava at the time of euthanasia.Kidney tissues were collected and cortex and medulla were separated bysharp dissection and flash frozen for analysis. Within a few minutesafter collection, blood samples were centrifuged at +4° C. and plasmawas separated and frozen. Urinary AVP levels were assayed by ELISA.Table 1 shows the groups of mice used in the studies

TABLE 1 Group ID Tica- Tica- Tica- Tica- Tica- Controls 0.05 0.10 0.150.20 0.25 Ticagrelor N/A 0.05% 0.10% 0.15% 0.20% 0.25% Dose in Food(wt/wt) Number of 6 5 5 5 5 5 Mice (N)DiscussionEffect of Different Doses of Ticagrelor on Urinary AVP, Urine Output andUrine Osmolality in Mice:

Different doses of ticagrelor (0.05 to 0.25%) were administered to miceembedded in standard rodent chow (w/w) for 14 days. Twenty-four hoururine samples were collected from the mice by placing them in individualplastic metabolic cages (1 mouse/cage) for two consecutive days, withfree access to food and water. Urine samples in the metabolic cages werecollected under a layer of light mineral oil. The volumes of urinesamples collected were recorded. Urine samples were centrifuged toremove particulate matter. Clear supernatants thus obtained were usedfor analysis. Blood samples were collected at the time of euthanasia onday 14, as per the protocol recommended by the AstraZeneca for samplesdestined for the determination of plasma ticagrelor. Briefly, underisoflourane anesthesia, blood from the posterior vena cava was collectedand transferred to EDTA K-coated microtubes (Microvette®, Sarstedt,Nümbrecht, Germany). Plasma was separated by centrifugation within 30min after collection and frozen at −80° C. The frozen samples wereshipped in dry ice by FedEx to AstraZeneca (Mölndal, Sweden). AtAstraZeneca R&D, the plasma concentration of ticagrelor was determinedby liquid chromatography tandem mass spectrometry (LC-MS/MS). FIGS. 1-5show the data obtained.

The data presented in FIGS. 1 through 5 clearly show that administrationof ticagrelor induces a dose-related decrease in urinary concentratingability in mice, as assessed by increase in urine output, associatedwith a corresponding decrease in urine osmolality. This decrease inurine concentrating ability is apparently due to a comparabledose-related decrease in urinary AVP excretion. Since AVP levels in24-hour collection of urine are a reliable surrogate for the circulatingplasma levels of AVP over the same period, it is logical to concludethat administration of ticagrelor results in dose-dependent decrease inAVP production by the hypothalamus. The modest alterations in urinaryparameters in the control group over time (from day 0 to 14) are oftenobserved apparently due to the stress associated with moving the micefrom regular to metabolic cages and vice versa. Their magnitude is farless than the effect seen with the increasing doses of ticagrelor.

It was discovered that administration of ticagrelor (Brilinta®) tonormal mice causes a dose-dependent decrease in urinary excretion ofarginine vasopressin (AVP), a reliable surrogate for circulating levelsof AVP. Circulating levels of AVP are critical for the cyst growth inADPKD. So, the therapeutic potential of ticagrelor in slowing down thecyst growth in ADPKD is currently being evaluated. This approach canhave minor side effects only as compared to the significant side effectswith other therapies under development.

AVP or the anti-diuretic hormone, elaborated by hypothalamus in thebrain, plays a critical role in body water homeostasis by regulating theurinary excretion of water. AVP, acting through its V2 receptor, is amajor stimulus for production of cyclic AMP (cAMP) in the kidneycollecting duct, the site of osmotic water reabsorption. However,collecting ducts are also the predominant site of the origin of cysts inthe kidney. cAMP also promotes cyst growth.

Incidentally, the circulating levels of AVP are increased in human ADPKDand in all animal models of ADPKD, where it has been ascertained. Thiscan be due to compensatory increase in the production of AVP byhypothalamus in response to reduced concentrating capacity of thepolycystic kidneys. Thus, AVP-V2 receptor-cAMP axis plays a criticalrole in ADPKD progression. Whatever may be the causative factors for theincreased AVP production, in pre-clinical studies in rodent models ofADPKD, vasopressin V2 receptor antagonists (OPC-31260 and tolvaptan)consistently inhibited cystogenesis by reducing cAMP production in thekidney. In subsequent clinical trials, the efficacy of tolvaptan for thetreatment of ADPKD was established.

ATP/UTP-activated P2Y2 and ADP-activated P2Y12 receptors were identifiedas potential targets for the treatment of acquired nephrogenic diabetesinsipidus (NDI) induced by lithium, a drug still widely used for thetreatment of bipolar disorder. Bipolar disorder has a high prevalenceamong war Veterans, because post-traumatic stress disorder (PTSD)predisposes war Veterans to bipolar disorder. P2Y12 receptor isexpressed in the kidney and hypothalamus, and clopidogrel bisulfate(Plavix®), an anti-clotting drug, which blocks platelet P2Y12 receptor,were found to significantly ameliorate lithium-induced NDI in rodentmodels. Ticagrelor is another P2Y12 receptor antagonist marketed asBrilinta®. Unlike clopidogrel, ticagrelor is not a pro-drug, and itreversibly binds to P2Y12 receptor, thus it has specific advantages overclopidogrel. It was determined that administration of ticagrelor tonormal mice (B6D2 genetic background) causes a dose-dependent decreasein the production of AVP as assessed by AVP levels in 24-hour urinecollections (a reliable surrogate for circulating levels of AVP over 24hours), associated with a dose-dependent decrease in urinaryconcentrating ability as assessed by urine output and osmolality, thusestablishing a reduced activity of AVP-V2 receptor-cAMP axis inticagrelor-treated mice (FIGS. 2-5). The ticagrelor-induced reduction inAVP-V2 receptor-cAMP can decrease cyst growth in ADPKD.

It should be noted that the effects shown in FIGS. 2-5 are unique toticagrelor, and could not be seen with either clopidogrel or withprasugrel (Effient®) another P2Y12 receptor antagonist. Althoughticagrelor does not pass through the blood brain barrier (BBB), and thushas limited exposure to brain, yet it can act on hypothalamus. It isbecause the hypothalamus or the posterior pituitary gland lacks a BBB,and thus is easily accessible to circulating drugs and agents. Hence,ticagrelor can have a direct effect on hypothalamus, and thus reduce AVPproduction. This effect of ticagrelor can also not be mediated throughP2Y12 receptor, because blockade of P2Y12 receptor in primary culturesof rat hypothalamic cells by PSB-0739, a potent and selectiveantagonist, actually increased the expression of AVP. Reducing AVPproduction in a controlled fashion (by adjusting the dose of ticagrelor)can have specific advantages over blockade of V2 receptor by the use ofselective receptor blockers for the treatment of ADPKD.

FIG. 4 confirms that significant reduction in urine concentratingability induced by 0.25% ticagrelor in the food was associated with amarked decrease in urinary AVP (vasopressin), a surrogate for thecirculating AVP levels. Furthermore, these data also reveal that lowerdoses of ticagrelor have minimal effect on urinary AVP. Note: The modestfall in urinary AVP in the control group over time (from day 0 to 14) isoften observed apparently due to the stress associated with moving themice from regular to metabolic cages and vice versa.

Further processing of data using regression analysis revealed asignificant relationship between plasma ticagrelor and urine osmolality(FIG. 5A) or urinary excretion of AVP (FIG. 5B) in the mice feddifferent concentrations of ticagrelor in the food. Similar to the datashown in FIGS. 3 and 4 for urine osmolality and AVP, respectively, therelation was more pronounced at plasma ticagrelor levels above 1.30 μM,corresponding to a dose of 0.15% or above. These data further confirmthat higher plasma concentrations of ticagrelor definitely result indecreased urine concentrating ability associated with lower AVP levels,whereas at lower concentrations of ticagrelor the effect is variable orhas less impact.

As, these data suggest that the observed dose-dependent decrease inurinary AVP is due to the administration of ticagrelor, which in turn istranslated to a dose-dependent decrease in urinary concentrating abilityas assessed by urine osmolality, the latter cannot be achieved withoutthe decreased activity of AVP-V2 receptor-cAMP axis in the renalcollecting duct cells as a function of plasma levels of ticagrelor. Inother words, ticagrelor has the potential to decrease cAMP production inthe renal collecting duct cells in a dose-dependent manner, which is thedesired effect for a candidate molecule to be developed as a drug forthe treatment of ADPKD.

Example 2

Primary cultures of rat inner medullary collecting duct (IMCD) cellswere prepared. When the cells became 70-80% confluent, the medium waschanged to relative starvation mode (low FBS), and used for testing theeffect of Desmopressin (dDAVP), a V2 receptor-selective analogue of AVP,and/or ticagrelor. Cells were incubated for 48 h with ticagrelor rangingfrom 1.5 to 25 μM. During the second 24 hours, cells were challengedwith dDAVP (20 nM). At the end of the incubation period, cells wereharvested, RNA extracted, reverse transcribed and subjected to real-timePCR to determine the expression of AQP2 and AQP3 genes relative to theexpression of the housekeeping genes (β-actin or GAPDH).

Results (FIGS. 6A-C) are presented as percent of the values in controlincubations without any addition of agents. FIGS. 6A-C show the effectof different concentrations of ticagrelor on dDAVP (20 nM)-induced AQP2or AQP3 expression relative to β-actin expression in primary cultures ofrat IMCD cells. Two independent assays on primary cultures of IMCD cellsderived from two different rats at different times were used, and thedata obtained were pooled for analysis and presentation in the graphs.Data were analyzed by analysis of variance (ANOVA) followed byTukey-Kramer Multiple Comparison Test. The numbers in parentheses abovethe bars indicate the number of culture wells. Ticagrelor showed atendency for concentration-dependent enhancement of the effect of dDAVPon AQP2 and AQP3 mRNA expression. The enhancements at 2 or 10 μM arestatistically significant (ANOVA followed by Tukey-Kramer MultipleComparison Test)

Blockade of P2Y12 receptor in IMCD cells by ticagrelor augmented thedDAVP-induced expression of both AQP2 and AQP3 genes. The augmentativeeffect was seen even at ticagrelor concentration of 2 μM, and is moreprominent at 10 μM. These findings are similar to the ones theinvestigators observed previously by the use of PSB-0739, a potent,selective and reversible antagonist of P2Y12 receptor. Thus, it appearsthat as expected, these effects of ticagrelor on IMCD cells are mediatedthrough P2Y12 receptor. However, at higher concentrations (25 μM),ticagrelor has a negative effect, suppressing the gene expression to avery low level. It should be noted that in general, for drugs that arefreely filtered at the glomeruli, and are not secreted by the tubules,the concentrations in the distal nephron or collecting duct in vivo areat least 10-fold higher than their plasma concentrations. So, 10 μM ofticagrelor used here in the culture dish is equivalent to 1 μM ofticagrelor in the plasma in vivo.

dDAVP acting through the vasopressin V2 receptor in IMCD cellsstimulates the production of cAMP (FIG. 7). In the collecting ducts invivo, in the short-term AVP increases cellular cAMP levels to causetranslocation of AQP2 protein from the subapical vesicles to apicalplasma membrane, and thus increase the water permeability of the apicalplasma membrane, the rate limiting barrier for transepithelial watertransport. In the long-term, increased cellular cAMP levels inducetranscriptional activation of AQP2 and other genes. Hence, theinvestigators evaluated the effect of ticagrelor on desmopressin-inducedcAMP production in primary cultures of rat IMCD cells. Primary culturesof rat IMCD cells were prepared and incubated with desmopressinwith/without ticagrelor (5 μM) as described above. For cAMP assay, themedium was gently aspirated, 200 μl of 0.1 M HCl was added to eachinsert and incubated for 20 min at room temperature. Then cells werescrapped off the semi-permeable support and lysed by pipetting up anddown. The cell lysate was centrifuged at 1,000×g for 10 min. cAMPconcentration in the supernatant was determined by an EIA kit (CaymanChemical Co.) and normalized to the protein content of the wells(Pierce™ BCA Protein Assay Kit, Rockford, Ill.).

Stimulation of IMCD cells with 20 nM of desmopressin caused 3.6-foldincrease in cellular cAMP levels. Addition of 5 μM ticagrelor did nothave any significant effect on desmopressin-induced cAMP levels. Thisfinding indicates that at the concentration used, ticagrelor does notinterfere with the action of desmopressin on the cAMP production in thecollecting duct. A higher concentration of ticagrelor (10 μM) can havean effect on the desmopressin-stimulated cAMP production in the IMCD.However, it should be noted that even at 2 μM concentration, ticagrelorshowed a tendency to augment the effect of dDAVP on AQP2 and AQP3expression. Furthermore, the concentration of ticagrelor used here (5μM) in cell cultures, when translated into in vivo conditions in anintact animal correspond to about 0.5 μM ticagrelor in blood plasma.Based on this, it appears that the observed in vivo effect of ticagrelorat low plasma concentrations on urinary osmolality is predominantly dueto low AVP levels, but not on the IMCD. It is because, in isolated IMCDcells, ticagrelor at a concentration of 5 μM did not suppressdDAVP-induced increases in cAMP production or APQ2/AQP3 expression.

Example 3

Based on the above observations in IMCD cells, where differentconcentrations of ticagrelor were used, it appears that at higherconcentrations, ticagrelor can have an effect on the IMCD cells ingeneral. At a concentration of 25 μM, ticagrelor almost completelysuppressed the gene expression of AQP2 and AQP3. So, the effect ofticagrelor on cell viability was investigated using two approaches asfollows (FIGS. 8A and 8B).

LDH Cytotoxicity Assay: Lactate dehydrogenase (LDH) is a cytosolicenzyme released into the cell culture medium when the plasma membrane isdamaged. So, quantification of extracellular LDH activity in the mediumindicates the viability of the cells exposed to agents. ThermoScientific™ Pierce™ LDH Cytotoxicity Assay Kit was used, which is asimple and reliable colorimetric method for quantifying cellulartoxicity. The kit is based on a coupled enzymatic reaction in which LDHcatalyzes the conversion of lactate to pyruvate via NAD+ reduction toNADH. Diaphorase then uses NADH to reduce a tetrazolium salt to redformazan product that can be measured at 490 nm. The level of formazanformation is directly proportional to the amount of LDH released intothe medium, which is indicative of cytotoxicity. Primary cultures of ratIMCD cells grown in collagen coated 96-well plate were incubated withdifferent concentrations of ticagrelor for 24 hours. LDH released intothe medium was assayed using the kit as per the manufacturer'sinstructions. Total LDH activity was determined by adding lysis bufferto the designated wells. Absorbance readings of the test wells wereconverted into the percent values in relation to the maximum LDHactivity. Results are presented in FIG. 8A.

Cell Cytotoxicity Assay: Toxicity of ticagrelor toward IMCD cells wascolorimetrically assayed using Cell Cytotoxicity Assay Kit from Abcam.This kit uses a proprietary water-soluble dye that changes itsabsorption spectra upon cellular reduction. The absorption ratio changeis directly proportional to the number of living cells on the plate.

According to the manufacturer, this assay has higher sensitivitycompared to the tetrazolium based colorimetric assays (such as MTT).Primary cultures of rat IMCD cells grown in collagen coated 96-wellplate were incubated with different concentrations of ticagrelor for 24hours. Cell cytotoxicity was assayed as per the manufacturer'sinstructions. Absorbance changes at 570 nm and 605 nm were monitored.The ratio of A570 and A605 were used to determine the cell viability ineach cell. Results are presented in FIG. 8B.

As compared to the currently available alternative for reduction in theactivity of AVP and production of cAMP in the kidney, i.e., vasopressinV2 receptor antagonism by tolvaptan, the disclosed invention hasspecific and clear advantages. First, administration of tolvaptan blocksthe vasopressin V2 receptor and thus causes severe polyuria (loss ofwater in the urine), nocturia (frequent urination in the night), thirstand dry mouth in a vast majority of the patients. Other common sideeffects of tolvaptan are constipation, loss of appetite, dry skin,nausea, vomiting, and potential for acute liver failure. Furthermore,tolvaptan is metabolized by the CYP3A4 system, which may result inincreased interactions with other medications. Although tolvaptanexhibits dose-related pharmacokinetics, it is unpredictable in ADPKDpatients (or animals) who have varying degrees of renal dysfunction,thus necessitating therapeutic drug monitoring. In view of this, thelong-term use of tolvaptan for the treatment of ADPKD is not feasible inmany patients. It should be noted that tolvaptan was originallydeveloped for the treatment of hyponatremia, which represents a shorterperiod of treatment. In contrast, the only major side effect oflong-term administration of ticagrelor is uncontrolled bleeding fromwound or internal ulcers. Other side effects reported are minor, andtolerable.

Besides, unlike tolvaptan, ticagrelor has been developed and evaluatedfor long-term use in patients with cardiovascular risk factors (e.g.,heart attack or stroke). Second, the ability of ticagrelor to reduce theproduction of AVP by the hypothalamus is an off-target effect, and isnot related to its original intended use to block P2Y12 receptorantagonist to function as a blood thinner. It should be noted that theinventors observed that clopidogrel bisulfate (Plavix®), anotherselective P2Y12 receptor blocker and blood thinner, did not have such aneffect on the production of AVP by the hypothalamus. In fact, theyshowed that direct inhibition of P2Y12 receptor in cultured cells byPSB-0739, a potent, selective and reversible antagonist, actuallyincreased the expression of AVP. At present the molecular mechanisms ofthe observed inhibitory effect of ticagrelor on the hypothalamus are notknown, thus making it difficult to mimic its action on the hypothalamusby other drug manufacturers. It takes considerable research to unravelthe molecular mechanisms by which ticagrelor inhibits the production ofAVP in the hypothalamus. In contrast, tolvaptan does not possess suchtacit mechanism of action, and its therapeutic effect both inhyponatremia and ADPKD is mediated through the same known mechanism,i.e., blockade of vasopressin V2 receptor, thus making it vulnerable tocompetition. Finally, it has been shown that chronic blockade ofvasopressin V2 receptor by tolvaptan results increased endogenousproduction of AVP. Since, AVP has two other receptors, namely, V1a (inblood vessels) and V1b (in liver), the effects of chronically increasedAVP levels through these two receptors is not known. In contrast,administration of ticagrelor decreases AVP levels in a controllablemanner, and so the V1a and V1b receptors are not overstimulated.

Discussion

The results obtained in these experiments have significant short- andlong-term impact on the treatment or management of ADPKD, the mostcommon form of PKD. In the short-term, the results offer a viabletreatment strategy for ADPKD with fewer or tolerable side effects.Ticagrelor (Brilinta®) has been in clinical use for about 5 years in theUnited States so ticagrelor can be directly tested in ADPKD patients forits therapeutic potential. Currently V2 receptor blockers, such astolvaptan, have been shown to slow down the cyst growth in experimentalmodels of ADPKD, and are undergoing clinical trials. The major sideeffect of long-term use of ticagrelor is uncontrolled bleeding fromwounds or internal ulcers. Other side effects reported for ticagrelorare minor and tolerable. Second, it has been shown that chronic blockadeof vasopressin V2 receptor by tolvaptan results in increased endogenousproduction of AVP. Since AVP interacts with two other receptors, namelyV1a (in blood vessels) and V1b (in liver), the effects of chronicallyincreased AVP levels through these two V1 receptor subtypes is notknown. In contrast, administration of ticagrelor decreases AVP levels ina dose-dependent fashion, and so the V1a and V1b receptors are notoverstimulated. Finally, in order to reduce renal cAMP levels, tolvaptanhas to swim against two opposing forces, namely increased levels of AVPand expression of V2 receptor in ADPKD. In contrast, by its potential toreduce circulating levels of AVP, ticagrelor essentially eliminates onecurrent, and thus reduces the availability of the ligand for theincreased V2 receptor expression in ADPKD.

Example 4

This study is proposed to evaluate the efficacy of ticagreloradministration in slowing down the growth of renal cysts in rodentmodels of ADPKD.

This study can also compare the efficacy of ticagrelor with tolvaptan inslowing down cyst growth, and on the major side effect, the polyuria. Itis expected that (i) administration of ticagrelor slows down renal cystgrowth, and (ii) the slowing down process is associated with minimal andtolerable loss of water in urine (polyuria) as compared to the use oftolvaptan. Thus, a new therapy for ADPKD can be developed using areadily available drug (ticagrelor), which has a proven safety recordand is well tolerated by patients on a chronic treatment regimen. Inaddition, administration of ticagrelor has the added benefit ofprevention of acute cardiovascular and cerebrovascular events (heartfailure and stroke) in high risk patients with ADPKD, a benefit notoffered by the use of V2 receptor blocks, such as tolvaptan.

The study design consists of evaluating the effect of administration ofticagrelor in mouse models of ADPKD. Syngeneic mice without ADPKD canserve as controls. One proposed ADPKD model uses mice lacking Pkd1 gene,which accounts for 85% of the human ADPKD patients. The other ADPKDmodel uses mutant mice lacking Pkd2 gene, which is responsible for 15%of prevalence of ADPKD in humans. Since germ line and/or whole bodydeletion of Pkd1 or Pkd2 can be lethal or the mice may or may notsurvive for long time due to rapidly progressing disease, inducible andrenal collecting duct specific knockout of Pkd1 or Pkd2 genes can beused. Cryopreserved embryos of Pkd1 or Pkd2 floxed mice are availablefrom the Jackson Laboratory, which rederives the mutant mouse lines andsupplies the breeders. The Jackson Laboratory also provides a variety ofCre-ER mice with different promoters and Nestin-CRE mice. For example,by crossing Pkd1-floxed mice or Pkd2-floxed mice with Nestin-CRE mice,“mosaic” model of ADPKD may be obtained. The “mosaic” ADPKD mouse modelis a slowly progressing PKD model, similar to the one seen in humanpatients, and thus allows us to test the effect of drugs. Theeffectiveness of ticagrelor as a therapeutic, used singularly or incombination with other drugs, for treating ADPKD and managing itssymptoms may be determined. In addition, the effectiveness of ticagrelorin comparison to vasopressin V2 receptor antagonists, such as tolvaptan,on the progression of ADPKD may be evaluated in these mutant mice.

i. To Establish Optimum Doses for Ticagrelor and Tolvaptan Vis-à-VisAVP-V2 Receptor-cAMP Axis.

The optimum dose of ticagrelor and tolvaptan can be determined toachieve a comparable level of activity of AVP-V2 receptor-cAMP axis, thekey determinant of cyst growth. A published report administeredtolvaptan concentrations between 0.01% and 0.30% in food in DBA/2FG-pcymouse model of ADPKD, and reported the optimum concentration as 0.10%.However, it is possible that each model of ADPKD may have its ownsensitivity toward tolvaptan. Similarly, as shown in FIGS. 1-4 and 5,ticagrelor concentrations between 0.05% and 0.25% in food were evaluatedon the urinary AVP excretion and concentration in normal mice, and awide range of effects were found. In view of the observed variations,dose-effects for both drugs can be evaluated by administering differentconcentrations in food and determining the level of activity of AVP-V2receptor-cAMP axis as follows. The “mosaic” ADPKD mouse model, describedabove, can be administered different concentrations of ticagrelor andtolvaptan in the food to control or wild type WT mice and mosaic Pkd1 ormosaic Pkd2 knockout mice. Table 2 gives the doses of ticagrelor andtolvaptan to be tested and the number of mice used for each dose.

TABLE 2 Proposed Doses and number of mice for each dose. TicagrelorDoses Tolvaptan Doses 0 0.10% 0.15% 0.20% 0.25% 0 0.03% 0.10% 0.20%0.30% WT N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 Pkd1 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 knockoutPkd 2 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5 N = 5knockout

The drugs will be administered mixed in the food. Treatment starts at 2to 3 months age and lasts for one month, at which point mice arehumanely euthanized. Blood and kidney samples can be collected foranalysis. Twenty-four hour urine samples can be collected prior to, atthe mid-point and towards the end of the treatment period. Urine can beanalyzed for output, osmolality and AVP content. Plasma samples can beanalyzed for ticagrelor. Kidney samples can be analyzed for theexpression of V2 receptor mRNA and cAMP levels, and AQP2 mRNA andprotein. The morphological structure of the collecting duct can beexamined for cyst development. Based on the data collected the doses ofticagrelor and tolvaptan that result in comparable levels of decrease incAMP levels in the kidney, and comparable levels of polyuria can bedetermined. However, from the point of therapeutic effect in ADPKD,comparable decreases in cAMP levels are more relevant than comparabledegree of decreases in polyuria. The data collected can also provideinformation on the level of AVP decrease in ticagrelor vs. increase intolvaptan-treated mice at the optimum doses selected. The optimum dosesof the two drugs that established can be used in the followingexperiments in Pkd1 and Pkd2 mosaic models of ADPKD.

ii. Comparative Effect of Ticagrelor and Tolvaptan on the Progression ofCyst Growth in Pkd1 or Pkd2 Mosaic Models of ADPKD.

After establishing optimum doses for ticagrelor and tolvaptan, groups ofmosaic Pkd 1 or Pkd 2 knockout mice and their WT (Pkd 1 floxed or Pkd 2floxed) mice can be subjected to treatment as shown in table 3. Cohortsof mosaic mutant mice and WT floxed mice do not receive any treatment(N=10 mice/group). Treatment with the drugs will start when the mice are2- to 3-month old and will continue until the time of euthanasia(approx. 6 to 7th month postnatal). Twenty-four hour urine samples willbe collected prior to the treatment, once in every month during thetreatment period and then towards the end of treatment.

TABLE 3 Proposed Doses and number of mice per treatment group TicagrelorTolvaptan 0 OPD* 0 OPD* Pkd 1 floxed N = 10 N = 10 N = 10 N = 10 Pkd 1knockout N = 10 N = 10 N = 10 N = 10 Pkd 2 floxed N = 10 N = 10 N = 10 N= 10 Pkd 2 knockout N = 10 N = 10 N = 10 N = 10 *Optimum DoseEstablished in earlier study

Blood and kidney samples can be collected at the time of euthanasia.Using standard parameters, the severity of structural and functionalaspects of ADPKD can be assessed in the treated and untreated mice andthe controls. The structural parameters are: i) body weight, ii) kidneyweight, iii) kidney/body weight ratio, iv) number of cysts per unit areaof the kidney under microscope, v) average size of the cysts in thekidney under the microscope, vi) cell proliferation in the kidney, andvii) apoptosis in the kidney. The functional parameters to be assessedare: i) urine output and osmolality, ii) urine AVP levels, iii) plasmachemistry, including blood urea nitrogen (BUN); v) eGFR (estimatedglomerular filtration rate), vii) analysis of kidney samples for theexpression of V2 receptor mRNA and cAMP levels, and AQP2 mRNA andprotein. The data collected thus can reveal the effects of ticagrelor ortolvaptan in Pkd1 and Pkd2 models of ADPKD when administered at theirrespective doses on: 1) severity of polyuria and associated urine andkidney parameters; ii) progression of ADPKD as assessed by number andsize of the cysts in the kidney; and iii) renal functional impairment,if any, as assessed by BUN and eGFR determinations. Thus, this studyevaluates the therapeutic potential of ticagrelor in ADPKD, as well asits side effects in comparison with that of tolvaptan.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

What is claimed is:
 1. A method for inhibiting arginine vasopressin(AVP) production in hypothalamus comprising administering tohypothalamic cells an effective amount of ticagrelor, thereby inhibitingarginine vasopressin (AVP) production in hypothalamic cells.
 2. Themethod of claim 1, further comprising administering one or moreadditional therapeutic.
 3. The method of claim 2, wherein the one ormore additional therapeutic is a mTOR inhibitor.
 4. The method of claim2, wherein the one or more additional therapeutic is a somatostatinanalogue.
 5. The method of claim 2, wherein the one or more additionaltherapeutic is a vasopressin V2 receptor antagonist.
 6. The method ofclaim 2, wherein the one or more additional therapeutic is an epidermalgrowth factor receptor inhibitor.
 7. The method of claim 1, wherein thedecrease in AVP production is determined by comparing AVP levelsdetected in the blood or urine of the subject before administeringticagrelor or its derivative to AVP levels detected in the blood orurine of the subject after administering ticagrelor or its derivative.8. The method of claim 1, wherein the effective amount of the ticagreloror derivative thereof is in a plasma range of about 1.0 to 6.0 μM.